AU2021255518A1

AU2021255518A1 - Recombinant fusion protein for preventing or treating fibrotic diseases

Info

Publication number: AU2021255518A1
Application number: AU2021255518A
Authority: AU
Inventors: Jun Seo Oh
Original assignee: Senelix Co Ltd
Current assignee: Senelix Co Ltd
Priority date: 2020-04-14
Filing date: 2021-03-31
Publication date: 2022-11-03
Also published as: KR102638021B1; KR20210154784A; WO2021210812A1; US20230357356A1; BR112022020350A2; CN115605502A; EP4137507A1; EP4137507A4; JP2023521906A; CA3175503A1

Abstract

The present invention relates to a fusion protein of albumin and a retinol-binding protein, which can be used to prevent or treat fibrotic diseases occurring in the liver, pancreas, lungs, and the like. The fusion protein of the present invention inhibits the activation of stellate cells, which causes tissue fibrosis, or induces the inactivation thereof, thus enabling the prevention or treatment of fibrotic diseases, and exhibits a more potent effect at a low concentration as compared to conventional fusion proteins for the same purpose. Thus, the fusion protein is expected to be a general-purpose base technology capable of remarkably reducing the dose and frequency of administration of protein therapeutic agents to the human body.

Description

[DESCRIPTION]

[Invention Title]

RECOMBINANT FUSION PROTEIN FOR PREVENTING OR TREATING FIBROTIC DISEASES

[Technical Field]

The present disclosure relates to a fusion protein of albumin and a

retinol-binding protein, which can be used to prevent or treat fibrotic diseases

occurring in the liver, pancreas, lungs, etc.

[Background Art]

Tissue fibrosis leads to a fatal result of the failure of tissue function. For

example, liver fibrosis results in the failure of liver function, followed by cirrhosis and

cancer, and fibrous tissues are observed in both chronic pancreatitis and pancreatic

cancer. No therapeutic agent for fibrotic diseases is available at present, and tissue

transplantation is employed as the most promising technique for treatment. The

molecular mechanism of tissue fibrosis has not been elucidated clearly yet, and

research and development are necessary for therapeutic agents for fibrotic diseases.

Recently, it was found out that tissue fibrosis occurs during the activation and

transdifferentiation of stellate cells that constitute tissues. Activated stellate cells

overexpress extracellular matrices such as collagen and differentiate into

myofibroblasts. The activated stellate cells play a decisive role in the onset of

tissue fibrosis.

Stellate cells are distributed not only in the liver but also in the pancreas, lungs, kidneys, intestines, etc. and play a central role in the regulation of retinoid homeostasis throughout the body. Food-derived vitamin A (retinol) circulates in the bloodstream as being bound to a retinol-binding protein (RBP) and is transported into stellate cells by the RBP receptor STRA6. Then, it is stored as retinyl ester in lipid droplets in the cytoplasm. Meanwhile, it is known that albumin inhibits the activation of stellate cells and the expression of albumin in activated stellate cells deactivates the cells.

In the previous research, the inventors of the present disclosure have

prepared a fusion protein which targets stellate cells, wherein RBP that can be

transported into stellate cells by STRA6 is bound to albumin, in order to inhibit the

activation of stellate cells or deactivated the cells and have identified its effect on the

prevention and treatment of fibrosis (KR 10-1395394).

[Disclosure]

[Technical Problem]

The present disclosure has been made to improve the effect of inhibiting the

activation of stellate cells or deactivating the cells of the existing RBP-albumin fusion

protein and is directed to providing an RBP-albumin fusion protein wherein one or

more amino acid sequence of albumin is substituted and RBP-albumin fusion

proteins with different arrangements.

The present disclosure is also directed to providing a method for producing

the recombinant protein of the present disclosure in large scale by providing a vector

capable of expressing the fusion protein and a transformant in which the vector is

introduced.

The present disclosure is also directed to providing a composition containing the fusion protein for preventing or treating a fibrotic disease since the fusion protein exhibits better effect of inhibiting the activation of stellate cells or deactivating the cells than the existing RBP-albumin fusion protein.

However, the technical problems to be solved by the present disclosure are

not limited to those mentioned above and other problems not mentioned above will

be clearly understood by those having ordinary skill in the art from the following

description.

[Technical Solution]

The present disclosure provides a fusion protein consisting of or containing

an amino acid sequence of SEQ ID NO 1 or 2.

The present disclosure also provides a fusion protein consisting of or

containing any amino acid sequence of SEQ ID NOS 3-8.

In an exemplary embodiment of the present disclosure, the fusion protein

may be for preventing or treating tissue fibrosis.

The present disclosure also provides a polynucleotide encoding the fusion

protein described above. The polynucleotide may consist of or contain any base

sequence of SEQ ID NOS 9-14.

The present disclosure also provides a recombinant vector containing a gene

encoding the fusion protein, and a transformant including the recombinant vector.

The present disclosure also provides a method for producing a fusion protein,

which includes: (1) a step of preparing a recombinant vector containing a gene

encoding a fusion protein containing any amino acid sequence of SEQ ID NOS 3-8;

(2) a step of preparing a transformant by introducing the recombinant vector into a

host cell; (3) a step of culturing the transformant; and (4) a step of obtaining a fusion protein from the cultured transformant.

In an exemplary embodiment of the present disclosure, the gene encoding

the fusion protein may contain a polynucleotide containing one or more base

sequence selected from a group consisting of SEQ ID NOS 9-14.

In another exemplary embodiment of the present disclosure, the host cell

may be a mammalian cell, specifically a mammalian cancer cell, more specifically a

CHO cell.

The present disclosure may also provide a pharmaceutical composition for

preventing or treating a fibrotic disease, which contains a fusion protein consisting of

any amino acid sequence of SEQ ID NOS 1-8.

The present disclosure may also provide a method for preventing or treating

a fibrotic disease, which includes a step of administering a fusion protein consisting

of any amino acid sequence of SEQ ID NOS 1-8 to a subject.

In an exemplary embodiment of the present disclosure, the subject may be a

human in need of prevention or treatment of a fibrotic disease, particularly a patient

with a non-alcoholic fatty liver disease.

In another exemplary embodiment of the present disclosure, the method may

further include a step of diagnosing the degree of fibrosis after the administration of

the fusion protein by conducting CT imaging or biopsy.

In an exemplary embodiment of the present disclosure, the fibrotic disease

may be one or more disease selected from a group consisting of liver fibrosis,

chronic hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated

steatohepatitis (CASH), lung fibrosis, renal fibrosis, renal failure, pancreatic fibrosis,

chronic pancreatitis and pancreatic cancer.

The present disclosure also provides a use of the fusion protein described above for preparation of a medication for preventing or treating a fibrotic disease.

[Advantageous Effects]

A fusion protein of the present disclosure inhibits the activation of stellate

cells, which causes tissue fibrosis, or induces the deactivation thereof, thus enabling

the prevention or treatment of fibrotic diseases, and exhibits a more potent effect at a

low concentration as compared to existing fusion proteins for the same purpose.

Thus, the fusion protein is expected to be a general-purpose platform technology

capable of remarkably reducing the dose and frequency of administration of protein

therapeutic agents to the human body.

[Brief Description of Drawings]

FIG. 1 shows a mechanism by which a fusion protein wherein albumin III is

bound to RBP acts as being included in an activated stellate cell.

FIG. 2 shows a result of comparing the effect of inducing the deactivation of

activated stellate cells of fusion proteins 1 and 2 of the present disclosure and

existing fusion proteins (RBP-albumin III and albumin III-RBP).

FIG. 3 shows a result of comparing the effect of inducing the deactivation of

activated stellate cells of fusion proteins 1 and 2 of the present disclosure with fusion

proteins 3-6.

FIG. 4A and FIG. 4B show a result of comparing the degree of liver fibrosis in

an animal model of liver fibrosis depending on the administration of fusion proteins 1

and 4 and RBP-albumin through sirius red staining.

[Best Mode]

Albumin is a multifunctional plasma protein synthesized primarily by liver

cells. Albumin has three domains, each formed by two subdomains A and B. It is

known that albumin binds to various hydrophobic substances including fatty acids

and retinoic acid and transports the molecules in blood. According to

crystallographic analysis, five strong fatty acid-binding sites are distributed

asymmetrically in albumin (one in subdomain IB, one between IA and IIA, two in IIIA,

and one inIIIB).

In the previous research, the inventors of the present disclosure have

prepared a fusion protein wherein albumin and a retinol-binding protein (RBP) for

targeting stellate cells are bound, and confirmed that, after being introduced into

activated stellate cells, the fusion protein deactivates the cells and thus enables

prevention and treatment of fibrotic diseases. In addition, the inventors of the

present disclosure have found out that retinoic acid (RA) plays an important role

during activation of stellate cells and the fusion protein controls the activation of

stellate cells by reducing the intracellular level of RA. Therefore, in order to develop

a fusion protein that can inhibit the activation of stellate cells or induce the

deactivation thereof through more stable binding to retinoic acid (RA), they have

prepared new albumin-RBP fusion proteins using sequence variations of albumin

and arrangements of albumin sub-domains and RBP of various combinations.

In the previous research, the domain III of albumin has shown the best effect

of inhibiting the activation of stellate cells or inducing the deactivation thereof in the

fusion proteins. Based on this result, the inventors of the present disclosure have

intended to prepare a recombinant fusion protein with enhanced binding ability to

retinoic acid.

Thus, presuming that the 526th and 600th bulky amino acids of domain III in

the existing fusion protein of albumin domain III and RBP interfere with binding to RA,

they have substituted them with smaller amino acids and investigated their effect of

deactivating activated stellate cells through binding with RA (see SEQ ID NOS 1 and

2).

Specifically, the inventors of the present disclosure have designed fusion

proteins 1 and 2 as follows by substituting the 526th amino acid residue

(phenylalanine) and the 600th amino acid residue (valine) of albumin domain III with

valine and alanine, respectively.

- Fusion protein 1: RBP (1-193) + albumin III {404-526(F--V)-600(V--A)-601}

- Fusion protein 2: albumin [signal peptide (1-18) + III

{404-526(F--V)-600(V-A)-601}] + RBP (17-192)

In addition, the inventors of the present disclosure have identified that the

sub-domains IB and IIIA of albumin bind very stably to retinoic acid and have

designed fusion proteins 3-6 as follows.

- Fusion protein 3: RBP (1-193) + albumin [IIA {404-517(V)} + IB (131-218)]

- Fusion protein 4: RBP (1-193) + albumin [IIA {404-517(V)} + IB (134-218)]

- Fusion protein 5: albumin [signal peptide (1-18) + IIIA {404-517(V)} + IB

(131-218)] + RBP (17-192)

- Fusion protein 6: albumin [signal peptide (1-18) + IIIA {404-517(V)} + IB

(134-218)] + RBP (17-192)

Then, the inventors of the present disclosure treated activated stellate cells

with the fusion protein 1 or 2 and compared the effect of deactivating the stellate

cells with previously developed fusion proteins (RBP-albumin III and albumin

III-RBP) by measuring the expression level of a-SMA (a-smooth muscle actin) and collagen type I, which are markers of activated stellate cells. As a result, it was confirmed that the fusion proteins 1 and 2 of the present disclosure can significantly reduce the expression of a-SMA and collagen I at lower concentrations as compared to the previously developed fusion proteins (see Example 1).

Through this, the inventors of the present disclosure have identified that the

substitution of the 526th and 600th amino acids of albumin III with amino acids of

smaller molecular weights can more effectively induce the deactivation of stellate

cells due to increased binding ability to retinoic acid.

Therefore, the fusion protein of the present disclosure wherein albumin III

with the 526th and 600th amino acids substituted independently and RBP are bound

can be used for treatment of fibrotic diseases. In the fusion protein, the amino acid

at the 526th amino acid residue of albumin III may be substituted with valine, alanine

or glycine, and the amino acid at the 600th amino acid residue may be substituted

with alanine or glycine. The fusion protein may consist of or contain an amino acid

sequence of SEQ ID NO 1 or 2.

Then, the inventors of the present disclosure compared the effect of inhibiting

the expression of a-SMA and collagen I of the fusion proteins 1 and 2, which showed

superior effect of inhibiting the activation of stellate cells as compared to the existing

fusion proteins, with fusion proteins 3-6. As a result, it was confirmed that the

fusion proteins 3-6 can effectively inhibit the activation of stellate cells at lower

concentrations (Example 2).

In addition, the inventors of the present disclosure investigated the effect of

ameliorating liver fibrosis of the fusion proteins 1 and 4 and the existing fusion

protein (RBP-albumin III) using a carbon tetrachloride-induced liver fibrosis animal

model. As a result, it was confirmed that the fusion proteins 1 and 4 can ameliorate fibrosis more effectively at lower concentrations as compared to the RBP-albumin III

(Example 3).

Therefore, the present disclosure may provide a pharmaceutical composition

for preventing or treating a fibrotic disease, which contains one or more fusion

protein selected from a group consisting of the fusion proteins 1-6, and may provide

a method for preventing or treating a fibrotic disease, which includes a step of

administering two or more fusion proteins selected from a group consisting of the

fusion proteins 1-6 to a subject.

Since it was confirmed that the fusion proteins 1-6 of the present disclosure

can induce the deactivation of activated stellate cells more effectively at lower

concentrations as compared to the existing RBP-albumin fusion proteins, it is

expected that the pharmaceutical composition of the present disclosure can be a

general-purpose platform technology capable of remarkably reducing the dose and

frequency of administration of protein therapeutic agents to the human body.

In the present disclosure, the "fibrotic disease" refers to a disease caused by

fibrosis whereby normal tissue is damaged and replaced by fibrous connective tissue,

resulting in malfunction of an organ, and includes any disease wherein a part of an

organ is hardened due to fibrosis without limitation, such as liver fibrosis, chronic

hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated steatohepatitis

(CASH), lung fibrosis, renal fibrosis, renal failure, pancreatic fibrosis, chronic

pancreatitis and pancreatic cancer.

In the present disclosure, the "subject" may be any mammal without limitation.

Specifically, it may be human or livestock.

In the present disclosure, prevention refers to any action of delaying the

onset of a fibrotic disease by administering the pharmaceutical composition according to the present disclosure, and treatment refers to any action of ameliorating or favorably changing the symptoms of a fibrotic disease by administering the pharmaceutical composition according to the present disclosure.

In the present disclosure, the albumin used to form the fusion protein may be

derived from any species. But, specifically, it may be one derived from the same

species as the subject to which it is administered in order to avoid the risk of

immunogenicity.

In the present disclosure, the pharmaceutical composition may further

contain, in addition to the fusion proteins 1-6, one or more substance known to be

capable of preventing or treating tissue fibrosis, and may further contain a suitable

carrier, excipient and diluent commonly used to prepare a pharmaceutical

composition.

In the present disclosure, the "carrier" is also called a vehicle and refers to a

compound that facilitates the delivery of a protein or a peptide into a cell or a tissue.

For example, dimethyl sulfoxide (DMSO) is a carrier commonly used to facilitate the

delivery of many organic substances into the cell or tissue of an organism.

In the present disclosure, the "diluent" is defined as a compound diluted in

water in which a protein or a peptide is dissolved in order to stabilize the biologically

active form of the protein or peptide. Salts dissolved in buffer solutions are used as

diluents in the art. Phosphate-buffered saline is a commonly used buffer solution,

because it mimics the salt state of human body fluid. Since the buffer salt can

control the pH of a solution at low concentration, the buffer diluent rarely modifies the

biological activity of a compound. Compounds including azelaic acid may be

administered to a human patient either as they are or in combination with other

ingredients or suitable carriers or excipients used in combination therapy.

In addition, the pharmaceutical composition according to the present

disclosure for preventing or treating a fibrotic disease may be formulated as a

powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a

formulation for external use such as an aerosol, etc. or a sterile injection solution

according to a conventional method. The composition of the present disclosure

may be administered orally or parenterally (e.g., intravenously, subcutaneously,

intraperitoneally or topically) depending on purposes. The administration dosage

will vary depending on the patient's condition and body weight, the severity of a

disease, the type of the formulation, administration route and administration period

but may be determined adequately by those skilled in the art. For example, about

0.001-1000 mg may be administered in combination with a pharmaceutically

acceptable carrier. The composition of the present disclosure may be administered

once or several times a day, if necessary, either alone or in combination with surgery,

hormone therapy, medication and use of biological response modifiers.

Meanwhile, a protecting group such as an acetyl group, a

fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group,

a stearyl group, polyethylene glycol (PEG), etc. may be bound to the amino terminal

of the recombinant fusion protein of the present disclosure, and the carboxyl terminal

of the peptide may be modified with a hydroxyl group (-OH), an amino group (-NH2),

an azide group (-NHNH2), etc.

In addition, a fatty acid, an oligosaccharide chain, nanoparticles (gold

particles, liposomes, heparin, hydrogels, etc.), an amino acid, a carrier protein, etc.

may be bound to the terminal or amino acid R-residue of the fusion protein of the

present disclosure. Such amino acid modification improves the potency and

stability of the protein of the present disclosure.

In the present disclosure, the term "stability" refers to not only in-vivo stability

but also storage stability (at room temperature or during storage in a freezer).

The present disclosure also provides a method for producing the fusion

protein described above, which includes:

(1) a step of preparing a recombinant vector containing a gene encoding a

fusion protein containing any amino acid sequence of SEQ ID NOS 3-8;

(2) a step of preparing a transformant by introducing the recombinant vector

into a host cell;

(3) a step of culturing the transformant; and

(4) a step of obtaining a fusion protein from the cultured transformant.

In the present disclosure, the "gene" should be understood as the broadest

meaning, which encodes a structural protein or a regulatory protein, and is not

included as long as it is a DNA fragment encoding the fusion proteins 1-6 of the

present disclosure. Specifically, it may contain any base sequence of SEQ ID NOS

9-14.

Also, in the present disclosure, the "vector" refers to a DNA construct

containing a DNA sequence which is operably linked to a suitable control sequence

capable of expressing a DNA in a suitable host. The vector may be a plasmid, a

phage particle or simply a potential genome insert. When transformed into a

suitable host, the vector may replicate and function independently of the host

genome, or may be integrated into the genome of the host in some cases. Because

a plasmid is the most commonly used type of a vector, the terms plasmid and vector

are used sometimes interchangeably in the present disclosure.

A base sequence is "operably linked" when it is placed into a functional

relationship with another base sequence. The base sequence may be a gene and a control sequence linked to be capable of expressing the gene when a suitable molecule (e.g., transcription-activating protein) binds to the control sequence. For example, a DNA for a pre-sequence or a secretory leader is operably linked to a

DNA for a polypeptide if it is expressed as a pro-protein which participates in the

secretion of the polypeptide; a promoter or an enhancer is operably linked to a

coding sequence if it affects the transcription of the sequence; or a ribosome-binding

site is operably linked to a coding sequence if it is positioned so as to facilitate

translation. Generally, the "operably linked" means that the DNA sequences being

linked are contiguous and, in the case of a secretory leader, contiguous and present

in a reading frame. However, an enhancer is not necessarily contiguous. The

linkage between the sequences is accomplished by ligation at convenient restriction

enzyme sites. If such sites do not exist, a synthetic oligonucleotide adaptor or a

linker is used in accordance with the conventional practice.

Also, in the present disclosure, "transformation" or "transfection" refers to the

introduction of a DNA into a host for replication as an extrachromosomal factor or by

chromosomal integration. In the present disclosure, the host cell used to prepare

the fusion protein is a CHO cell, although not being limited thereto.

[Mode for Invention]

The present disclosure may be changed variously and may have various

exemplary embodiments. Hereinafter, specific exemplary embodiments will be

presented and described in detail. However, the present disclosure is not limited to

the specific exemplary embodiments but should be understood to include all

changes, equivalents and substitutes encompassed in the technical idea and scope

of the present disclosure. In the following description of the present disclosure, details of known technologies may be omitted to avoid unnecessarily obscuring the present disclosure.

[Experimental methods]

1. Isolation and culturing of hepatic stellate cells (HSCs)

After perfusing the liver of 14-week-old male BALB/c mice with PBS

(phosphate-buffered saline), followed by perfusion with GBSS (Gey's balanced salt

solution) containing collagenase, pronase and DNase, the liver was extracted. After

removing gall bladder and connective tissues attached to the liver and then placing a

suspension of the hepatic cells in the same GBSS and treating at 37 °C for 12

minutes, centrifugation was performed at 1400 g for 20 minutes in a 13.4%

Nycodenz gradient. Stellate cells were taken from the interface between the

Nycodenz solution and an aqueous layer and cultured in DMEM (Dulbecco's

modified Eagle's medium; Carlsbad, CA) supplemented with 10% fetal bovine serum

(FBS). The purity of the stellate cells was evaluated by microscopic observation

and western blotting using anti-tyrosine aminotransferase antibodies. When the

cells were confluent, they were subcultured and used as activated stellate cells.

The activation of the hepatic stellate cells was confirmed by morphological change

and increased expression of a-SMA and collagen I.

2. Design and preparation of fusion protein

2-1. Design of fusion protein

Detailed information about fusion proteins 1-6 for enhancing binding ability to

retinoic acid is shown in Table 1. The amino acids represented by lowercase letters

indicate the sequence of a signal peptide, and the amino acids substituted in albumin

III are bold-faced and underlined, and the underlined amino acids indicate RBP.

[Table 1]

SEQ Fusion protein Amino acid sequence ID NO

mkwvwaIllaawaaaERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFL

Fusion protein QDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKF

1: RBP (1-193) KMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSF

+ albumin III VFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNL 3 {404-526(F->V IKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKC

)-600(V->A)-6 CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP

01} CFSALEVDETYVPKEVNAETFTFHADICTLSEKERQIKKQTALVELVKHKP

KATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLAA

Fusion protein mkwvtfisllflfssaysLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEV

2: albumin STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTP

[signal peptide VSDRVTKCCTE SLVNRRPCFSALEVDETYVPKEVNAETFTFHADICTLSEK

(1-18)+ Ill ERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA 4 {404-526(F-> EEGKKLAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNI

V)-600(V->A)- VAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMK

601}] + RBP YWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFS

(17-192) RDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG

MkwvwalIllaawaaaERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFL

QDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKF Fusion protein KMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSF 3: RBP (1-193) VFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNL 5 + albumin [IIIA IKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKC {404-517(V)} +

CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP IB (131-218)] CFSALEVDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYF

YAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA

6 Fusion protein MkwvwallllaawaaaERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFL

4: RBP (1-193) QDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKF

+ albumin [IIIA KMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSF

{404-517(V)}+ VFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNL

IB (134-218)] IKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKC

CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP CFSALEVPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA

mkwvtfisllflfssaysLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYT Fusion protein KKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLC 5: albumin VLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDDNPNLPRLVRPEVDVM

[signal peptide CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK 7 (1-18) + IIIA AACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAK {404-517(V)} +

KDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFT IB (131-218)] +

DTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDG RBP (17-192) TCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG

mkwvtfisllflfssaysLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEV Fusion protein STPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTP 6: albumin VSDRVTKCCTESLVNRRPCFSALEVPNLPRLVRPEVDVMCTAFHDNEETF

[signal peptide LKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDEL 8 (1-18) + IIIA RDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQD {404-517(V)} +

NIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKM IB (134-218)] +

KYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVF RBP (17-192) SRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG

2-2. Preparation of fusion protein

After preparing polynucleotides encoding the designed fusion proteins 1-6,

recombinant expression vectors were prepared by cloning each DNA fragment into a

Xbal/Kpnl cut pcDNA3.1(+) vector. Detailed information of the DNA fragments that encode the fusion proteins is summarized in Table 2. Transformants expressing the fusion proteins were prepared by introducing each vector into CHO cells using

Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The fusion proteins expressed and

secreted by culturing the transformants were used after purifying by affinity

chromatography and HPLC.

[Table 2]

Fusion SEQ protein-encoding Sequence ID NO polynucleotide

CCGCTCTGGTGGAGCTGGTGAAGCATAAGCCTAAGGCTACAAA 9 Fusion protein 1 GGAGCAGCTGAAGGCCGTGATGGACGATTTCGCTGCCTTTGTG

GAGAAGTGCTGTAAGGCTGACGATAAGGAGACCTGTTTCGCCG AGGAGGGCAAGAAGCTGGCGGCT TGGATGGCACCTGTGCCGACAGCTATTCTTTCGTGTTTTCCCGC

GATCCTAATGGCCTGCCCCCTGAGGCTCAGAAGATCGTGAGGC 10 Fusion protein 2 AGAGGCAGGAGGAGCTGTGCCTGGCCAGGCAGTACCGGCTGA

TCGTGCATAATGGCTATTGTGACGGC

atgagatcgccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtaca

11 Fusion protein 3 aggctgccttcacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctgga

tgagctgagggacgagggcaaggcttcttccgcc

ccagacgccatccctacttttatgctcctgagctgctgttctttgccaagcggtacaaggctgcctt

12 Fusion protein 4 cacagagtgctgtcaggctgctgacaaggctgcttgcctgctgccaaagctggatgagctgag

ggacgagggcaaggcttcttccgcc

GACTGCTGAACCTGGATGGCACATGTGCTGACTCTTATTCCTTC

GTGTTTTCTCGCGATCCAAATGGCCTGCCCCCTGAGGCCCAGA 13 Fusion protein 5 AGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGC

AGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGC ACCTGGATGGCACATGTGCTGACTCTTATTCCTTCGTGTTTTCTC

GCGATCCAAATGGCCTGCCCCCTGAGGCCCAGAAGATCGTGAG 14 Fusion protein 6 GCAGAGGCAGGAGGAGCTGTGCCTGGCTAGGCAGTACCGGCT

GATCGTGCATAATGGCTATTGTGACGGC

3. Quantitative real-time PCR

Total RNAs were prepared using TRIzol (Ambion, Austin, TX, USA) and

cDNAs were constructed. qRT-PCR was conducted using the ABI QuantStudioTM 3

real-time PCR system. The PCR products were normalized to the mRNA level of

GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

4. Preparation of liver fibrosis animal model

A mouse model of liver fibrosis was prepared by inducing liver damage by

intraperitoneally injecting CC14 dissolved in mineral oil at 1:1 to BALB/c mice at a

concentration of 1 mL/kg for 6 weeks, 3 times a week. Only mineral oil of the same

amount was administered to a control group. The mice were sacrificed 48 hours

after the final injection of the CCl4.

For evaluation of the fibrosis of the sacrificed mice, liver was taken out and

tissue sections were prepared by fixing the liver tissue of each test group in 10%

buffered formalin and embedding in paraffin. For histological analysis, the tissue

sections were stained with sirius red and then observed with an optical microscope.

5. Statistical analysis

The results were expressed as mean standard deviation (SD). Statistical

analysis was conducted by t-test. P < 0.05 was considered significant.

[Experimentalresult]

Example 1. Comparison of effect of fusion proteins 1 and 2 with

existing albumin-RBP fusion proteins

The effect of the fusion proteins 1 and 2 designed in Example 2 was

compared with that of RBP-albumin III and albumin III-RBP, which showed superior

effect of inhibiting the activation of stellate cells or deactivating the same in the

previous research. Specifically, after treating activated mouse hepatic stellate cells

with 0.25 pM of each fusion protein for 20 hours, the expression level of a-SMA and

collagen I was measured by real-time PCR. The existing fusion proteins

(RBP-albumin III and albumin III-RBP) are the R-III and III-R fusion proteins of KR

10-1395394.

As a result, it was confirmed that the fusion proteins 1 and 2 decreased the

mRNA expression of a-SMA and collagen I as compared to the existing fusion

proteins, as shown in FIG. 2.

Example 2. Comparison of effect of fusion proteins 1-6

The effect of the fusion proteins 1 and 2, which showed superior effect of

deactivating activated stellate cells as compared to the existing fusion proteins, was

compared with that of the fusion proteins 3-6. Activated stellate cells were treated

with the fusion proteins in the same manner as in Example 1 at a concentration of

0.125 pM.

As a result, it was confirmed that the fusion proteins 3-6 can significantly

reduce the expression of a-SMA and collagen I with smaller protein contents, as

shown in FIG. 3.

Example 3. Comparison of effect of fusion proteins 1 and 4 with

existing albumin-RBP fusion proteins in vivo

The therapeutic effect of the fusion proteins was compared using a liver

fibrosis induced by carbon tetrachloride (CCl4) injection. Specifically, after

intraperitoneally injecting carbon tetrachloride dissolved in mineral oil at 1:1 to

BALB/c mice at a concentration of 1 mL/kg for 6 weeks, 3 times a week, the fusion

protein (25, 12.5, 6.25 or 3 pg) or saline was intravenously administered for 2 weeks

after the final injection of the CCl4, 3 times a week (n = 5).

As a result of observing tissues with an optical microscope, the mice of a

control group to which only mineral oil was administered showed normal liver

structure, whereas the mice treated with CC14 showed severe liver tissue damage

and fibrosis. In addition, it was confirmed that the administration of the

albumin-RBP fusion proteins resulted in alleviation of fibrosis (FIG. 4A).

As a result of quantitative analysis of siruis red staining using the ImageJ

software (NIH), the effect of alleviating fibrosis was different depending on the

samples (FIG. 4B). Whereas the existing fusion protein RBP-albumin III showed

significant effect only in the 25 pg administration group, the fusion protein 4 showed

superior effect also in the 6.25 pg administration group. From this result, it was

confirmed that the fusion proteins 1 and 4 exhibit superior effect at low

concentrations as compared to the existing fusion proteins.

While the specific exemplary embodiments of the present disclosure have

been described in detail, it will be obvious to those having ordinary knowledge in the

art that they are merely specific exemplary embodiments and the scope of the present disclosure is not limited by them. Accordingly, it is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

[Industrial Applicability]

A fusion protein of the present disclosure is expected to be a

general-purpose platform technology capable of remarkably reducing the dose and

frequency of administration of protein therapeutic agents to the human body.

Claims

[CLAIMS]

[Claim 1]

A fusion protein comprising an amino acid sequence of SEQ ID NO 1 or 2.
[Claim 2]

A fusion protein comprising any amino acid sequence of SEQ ID NOS 3-8.
[Claim 3]

A polynucleotide encoding the fusion protein of claim 2, wherein the

polynucleotide comprises any base sequence of SEQ ID NOS 9-14.
[Claim 4]

A recombinant vector comprising the polynucleotide of claim 3.
[Claim 5]

A transformant comprising the recombinant vector of claim 4.
[Claim 6]

A method for producing a fusion protein, comprising:

(1) a step of preparing a recombinant vector comprising a gene encoding a

fusion protein comprising any amino acid sequence of SEQ ID NOS 3-8;

(2) a step of preparing a transformant by introducing the recombinant vector

into a host cell;

(3) a step of culturing the transformant; and

(4) a step of obtaining a fusion protein from the cultured transformant.
[Claim 7]

A pharmaceutical composition for preventing or treating a fibrotic disease,

comprising a fusion protein comprising any amino acid sequence of SEQ ID NOS

1-8.
[Claim 8]

The pharmaceutical composition according to claim 7, wherein the fibrotic

disease is one or more disease selected from a group consisting of liver fibrosis,

chronic hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated

steatohepatitis (CASH), lung fibrosis, renal fibrosis, renal failure, pancreatic fibrosis,

chronic pancreatitis and pancreatic cancer.
[Claim 9]

A method for preventing or treating a fibrotic disease, comprising a step of

administering a fusion protein comprising any amino acid sequence of SEQ ID NOS

1-8 to a subject.
[Claim 10]

A use of a fusion protein comprising any amino acid sequence of SEQ ID

NOS 1-8 for preparation of a medication for preventing or treating a fibrotic disease.